Bruch Membrane

Segmentation of retinal layer boundaries on each OCT retinal B-scan and thickness maps of the desired retinal cell layers were performed and generated by Heidelberg software (Fig. 3C). Internal limiting membrane (ILM), external limiting membrane (ELM) and basement membrane (BM refers to the basement membrane of the choriocapillaries/Bruch’s membrane) were used for segmentation. The Heidelberg Spectralis segmentation software reliably detects these structures and provides accurate segmentation. Given that previous studies have shown significant thickness changes in the outer retinal layer, for the thickness maps, the retinal cell layers chosen for analysis were the outer retinal layer (between BM and ELM), inner retinal layer (between ELM and ILM), and overall retina (between BM and ILM).
For each analyzed retinal layer, the thicknesses across the 49 scans were averaged across nine regions of interest, including the foveal and the inner and outer perifoveal (superior, nasal, inferior, and temporal) regions (Fig. 1A, Supplementary Figs. 1–4A).
Averaged thicknesses across the foveal and inner and outer perifoveal regions were compared before and after dark adaptation for each subject. The absolute values of the before-and-after differences were compared between dark adapted/patched versus unpatched control eyes by paired t testing, two-tailed. Statistical significance was defined as p < 0.05.
For band intensity evaluation, all B-scans for the dark adapted/patched and unpatched control eyes, before and after the dark adaptation period, were masked and quantitatively and assessed for their EZ and IZ band intensities.
For assessing EZ band intensity changes, we used EZ:ELM ratio to eliminate potential intensity changes introduced to each scan because of factors such as media clarity, head tilt etc. Invitrogen GelQuant Express software was used to evaluate EZ band intensity changes over the central 2600 µm region on the central line scan. This region was split into 11 boxes, and due to irregular contour of bands in the fovea, the central-most box was excluded from analysis. The EZ and ELM band intensities were measured and the EZ:ELM ratios were calculated and averaged across the remaining 10 boxes. The EZ:ELM ratios, after vs before the dark adaptation period, were compared between light and dark adaptation conditions using paired t-testing, two-tailed.
In addition to EZ band intensity, we evaluated OCT images for any clinically significant changes. In a pilot observation we noticed some changes in the length of visibility of IZ band before and after dark adaptation. Thereafter, the IZ band lengths, before and after the dark adaptation period, were compared using paired t-testing, two-tailed.