Reader program

For OCT images captured by Bioptigen Envisu system, averaged radial scan images were used for retinal thickness measurement. For each eye, measurements were performed on four spots (450 μm from center of ONH at both horizontal and vertical directions) using vendor provided Reader program (Bioptigen), and the averaged number was used as the measurement for the eye. Total retina thickness was measured from nerve fiber layer (NFL) to basal side of RPE layer (Bruch's membrane [BM]). Outer retina length was measured from outer limiting membrane (OLM) to BM, two clearly distinguishable OCT layers even for degenerative retina. The same four spots were also used to calculate intensity profile across the outer retina based on averaged value of 10-pixel width at the spot center of OCT images. The width of EZ (IS/OS) (the inner segment ellipsoid zone, also referred as the inner segment/outer segment [IS/OS] junction^{10 (link),25 (link)–27 (link, link)}) was calculated from mid-point of ascending and descending phases on intensity profile of the band (as illustrated in Fig. 1). For OCT images captured with Heidelberg Spectralis, the frame of OCT B-scan that showed the largest subretinal fluid accumulation from each eye was selected for analysis. The highest values of outer retina length (from OLM to BM) were measured. Data are presented as mean ± SEM.

The procedure used for OCT imaging of mouse retina followed a published protocol.^{5 (link),27 (link)} Briefly, after mice were anesthetized with ketamine (100 mg/kg) and xylazine (6 mg/kg), retina OCT images were captured with Envisu UHR2200 (Bioptigen, Durham, NC, USA). The mouse eye was positioned with the ONH in the center of the OCT scan. Full field volume scans (1.4 mm × 1.4 mm at 1000 A-scan × 100 B-scan × 5) and two radial scans (at horizontal and vertical position and averaged 40 times) were captured. Mice retina were first imaged after ∼20 minutes of adaptation to room light (500 lux) in the procedure room under standard illumination conditions, and again in darkness after overnight dark adaptation. Averaged radial scan images were used for retinal thickness measurement. For each eye, measurements were performed on 4 spots (450 μm from center of ONH at both horizontal and vertical directions) by using the vendor-provided Reader program (Bioptigen), and an averaged number was used as the measurement for the eye. Outer retina length was measured from the outer limiting membrane to the RPE-choroid boundary.

Light exposure elicits an increase in OR thickness as measured by OCT in mice and humans; the procedure has been previously published.22 (link)–24 (link, link),26 (link) Briefly, after anesthetizing mice with ketamine (100 mg/kg) and xylazine (6 mg/kg), retina OCT images were captured with Envisu UHR2200 (Bioptigen, Durham, NC, USA), with OCT beam bandwidth of 160 nm, and theoretic axial resolution of 1.6 μm in tissue. The mouse eye was positioned with the optic nerve head (ONH) in the center of the OCT scan. Full-field (50° fixed field view, corresponding to 1.4 mm × 1.4 mm for a typical mouse eye) volume scans (at 1000 A-scan × 100 B-scan × 5) and a vertical B-scan (averaged 40 times) were collected. Mice used in this study were of similar age, so between-mice eye size differences were small. Vertical B-scan images were studied from our previous results showing that d-cis-diltiazem produces oxidative stress in superior and inferior retina,9 (link) and OR thickness was measured at location ∼450 μm superior (“12-o'clock” position) and ∼450 μm inferior (“6-o'clock” position) to the center of the ONH, by using vendor-provided Reader program (Bioptigen) and an in-house MATLAB program. OR length was measured from external limiting membrane (ELM) to the RPE-choroid boundary.