Subjects were imaged using the commercially available spectral-domain OCT (RTVue XR Avanti; Optovue, Inc., Fremont, CA, USA) with AngioVue software to obtain en face OCT angiograms as previously described.
10 The Avanti OCT provides 70,000 A-scans/second to acquire OCT angiograms consisting of 304 × 304 A-scans. Each OCT angiogram was created using orthogonal registration and merging of two consecutive scan volumes. Thirteen OCT angiograms (3 × 3 mm) were obtained using the Angio Retina mode in adjacent regions of the posterior pole by moving the software scanning area without changing fixation (
Fig. 1), which was a modification of the method of de Carlo et al.
11 Two scans of the superior and inferior optic disc were obtained using the Angio Disc mode. The retina has multiple vascular planes.
6,13 The RPC network is the most superficial of the retinal vasculature and courses parallel to the nerve fibers. The ganglion cell layer has one or more capillary layers, which were referred to as the “inner retinal plexus” by Spaide et al.
6 The capillaries of the inner retinal plexus under the RPC network connected to the small branches of the retinal arterioles and venules, and their spread is obviously not in parallel with the nerve fibers. To depict the RPC network and minimize inclusion of the capillaries of the inner retinal plexus, the segmentation of the inner border was fixed at the vitreous cavity, and that of the outer border was moved down manually from the internal limiting membrane (ILM) toward the RNFL in the cross-sectional OCT B-scan with overlaying blood flow. The outer boundary on each OCT angiogram was determined at the layer that was just above the layer depicting the small branches of the retinal arterioles and venules that connect the capillaries of the inner retinal plexus (
Fig. 2). The average depth of the outer border (in μm) from the ILM in each region to depict the RPC was calculated. ImageJ software (version 2.0.0-rc-41/1.50d; National Institutes of Health, Bethesda, MD, USA) with the plug-in MosaicJ (
http://bigwww.epfl.ch/thevenaz/mosaicj/; in the public domain) then was used to automatically assemble the 3 × 3-mm OCT angiograms to create one wide-field montage OCT angiogram of the RPC network. Two graders (TM, AI) performed three measurements of the maximal distance (in mm) in the superior and inferior temporal retina from the edge of the optic disc and the minimal distance (in mm) from the fovea in which the RPC was observed on each montage OCT angiogram. The data were averaged, and the intraclass correlation coefficient (ICC) was calculated to assess the interexaminer reproducibility for both distance measurements. In all 20 enrolled eyes, three of the first and second branches of the retinal arterioles were manually selected on each montage OCT angiogram to measure the capillary-free zone (CFZ). The arteriolar branches that were not seen entirely on the OCT angiograms and/or side by side with the venules were not measured. The areas (mm
2) of the CFZ beside the first and second branches of the retinal arterioles in the 0.5 × 0.5-mm squares were measured. The areas of the CFZ obtained by two graders (TM, AI) were averaged.