Retinal Vascular and Cellular Morphometry

ADPase-stained retinal flatmounts were used to determine the tortuosity index and vessel diameter. For the tortuosity index, a line was traced along the tortuous arteries using the polyline tool compared to a straight line traced from the vessel origin at the optic disk to the branch point using the arbitrary line tool of the CellSens software (Olympus America Inc., Center Valley, PA, USA) as previously described [33 (link),34 (link),60 (link),61 (link)]. Vessel diameter at the optic disk was quantified using the arbitrary line tool of the CellSens Dimension software (Olympus America Inc., Center Valley, PA, USA). The number of endothelial cells present in the nerve fiber layer (NFL)/ganglion cell layer (GCL), the total retinal thickness, and the thickness of the NFL/GCL, inner plexiform layer (IPL), INL (inner nuclear layer), and ONL (outer nuclear layer) were quantified in the H&E stained sections using the count and measure of region of interest and arbitrary line tools of CellSens Dimension software (Olympus America Inc., Center Valley, PA, USA).