In vivo characterization of the spatial-temporal distribution of erythrocytes in human retinal capillaries
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To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina.Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc.We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm(2) of retinal area).We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease.
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Retinal vasculature develops in a highly orchestrated three-dimensional (3-D) sequence. The stages of retinal vascularization are highly susceptible to oxygen perturbations. We demonstrate that optical tissue clearing of intact rat retinas and light-sheet microscopy provides rapid 3-D characterization of vascular complexity during retinal development. Compared with flat mount preparations that dissect the retina and primarily image the outermost vascular layers, intact cleared retinas imaged using light-sheet fluorescence microscopy display changes in the 3-D retinal vasculature rapidly without the need for point scanning techniques. Using a severe model of retinal vascular disruption, we demonstrate that a simple metric based on Sholl analysis captures the vascular changes observed during retinal development in 3-D. Taken together, these results provide a methodology for rapidly quantifying the 3-D development of the entire rodent retinal vasculature.
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Purpose To characterize human parafoveal blood flow using adaptive optics scanning laser ophthalmoscopy (AO-SLO). Methods In 5 normal subjects, erythrocyte aggregate distributions were analyzed on 3 different days. Erythrocyte aggregates were described as a “dark tail” in AO-SLO. The characteristics of the pathways with dark tail flow in the parafovea were measured. Additionally, the tendency for dark tail flow before and after bifurcations was analyzed to study the blood flow in detail. Results Average velocity in parent vessels with dark tail flow was 1.30±0.27 mm/s. Average velocity in daughter vessels with dark tail flow was 1.12±0.25 mm/s, and the average velocity of plasma gaps in daughter vessels without dark tail flow was 0.64±0.11 mm/s. Downstream from the bifurcations, the velocity in vessels with dark tail flow was higher than that in those without it (p<0.001), and the branching angles of vessels with dark tail flow were smaller than those of vessels without it (p<0.001). Conclusions Images from the AO-SLO noninvasively revealed pathways with and without dark tail flow in the human parafovea. Pathways with dark tail flow in the daughter vessels generally had faster flow and smaller bifurcation angles than daughter vessels without dark tail flow. Thus, AO-SLO is an instructive tool for analyzing retinal microcirculatory hemodynamics.
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