Insight software

Spectral domain OCT with the guidance of a bright-field live fundus image was performed with the image-guided OCT system (Phoenix Research Labs, Pleasanton, CA, USA) according to the manufacturer’s instructions and using the StreamPix 6 software version 7.2.4.2 (Phoenix Research Labs, Pleasanton, CA, USA) to generate fundus images and OCT scans. Using the InSight software, version 2.1.7237, (Phoenix Research Labs, Pleasanton, CA, USA), the borderlines between the retinal layers were defined on the OCT pictures. These borderlines were initially indicated automatically by the software; they were then manually corrected by the researchers, when necessary. Next, the distance (in µm) between each borderline was calculated using the InSight software at 300 consecutive points throughout the borderline, and the average of these data was defined as the thickness of the respective layer (NFL + IPL, INL, OPL, ONL, outer+ inner segments, RPE, and Choroid).

In vivo analysis of the retina was carried out using Micron IV together with Image-Guided 830 nm OCT (Phoenix Research Laboratories, Pleasanton, CA, United States). The animals were anesthetized with intraperitoneal injection of 80 mg/kg Ketamine, 10 mg/kg Xylazine (Sigma-Aldrich, Munich, Germany). OCT scans were acquired in mydriatic animals through a bidimensional scan (B-scan), performing a 550 μm diameter circular scan around the optic nerve head. Both eyes were examined and results were averaged. Retinal layer segmentation and quantification was performed using Insight software (Phoenix Research Laboratories). Results shown here concern the sum of the thicknesses of Retinal Nerve Fiber Layer (RNFL, that contains RGC axons) plus Ganglion Cell Layer (GCL, that contains RGC bodies). The measurement of RNFL/GCL complex in the mouse is preferable to the measurement of RNFL alone that is usually too thin to be correctly quantified (Jagodzinska et al., 2017 (link)).

SD-OCT (Micron IV; Phoenix Research Laboratories, Pleasanton, CA, USA) was used to measure the changes in GCC thickness before and three weeks after ONC injury, as previously described [2 (link), 43 (link)]. Briefly, radial volume scans (centered on the optic disc, with a diameter of 1.2 mm) were performed, and each volume consisted of 100 B-scans with 1000 A-scans per B-scan. Four images (scans 1, 26, 51, and 76 at 0°, 45°, 90°, and 135° in en face images) were analyzed using InSight software (version 1.1.5207, Phoenix Research Laboratories) and used for retinal thickness measurements. For each selected image, a vertical caliper was placed on each side of the optic nerve head (ONH) 500 µm away from the center of the ONH. The caliper was used to measure the thickness of the GCC, which consisted of the three innermost retinal layers: the NFL, GCL, and IPL. The GCC thickness of each retina was taken as the average of a total of eight measurements. The image analysis was performed in a blinded manner.

OCT was used to measure inner retinal thickness as previously described [20 ]. Briefly, fundus and OCT images of the retina around the optic disc were obtained, and the Insight software (Phoenix Research Labs, USA) was used to measure the retinal thickness.

Image-guided spectral-domain OCT images were taken using inSight software (Phoenix Research Labs). Genteal was applied to the eyes before imaging to keep cornea moist. Trans-retinal images were taken from regions around the optic nerve. Retinal thickness was calculated in the software.

Optical coherence tomography (OCT) and fluorescein angiography (FA) were performed as previously described (Buccarello et al., 2017 (link)), taking advantage of the Micron IV instrument (Phoenix Research Laboratories, Pleasanton, CA, United States). Briefly, after anesthesia, mydriasis was induced by administering a drop of tropicamide 0.5% (Visumidriatic, Tibilux Pharma, Milan, Italy) in each eye. OCT images were acquired by performing a circular scan of 550 μm of diameter around the optic nerve head. Both eyes were examined, and the results were averaged. The segmentation of retinal layers was performed using Insight software (Phoenix Research Laboratories, Pleasanton, CA, United States), OCT was followed by the FA study. A solution of 1% fluorescein (5 ml/kg Monico S.p.A., Venezia, Italy) was administered by a single intraperitoneal injection (100 μL). For each animal, the images of central and peripheral retinal vasculature were acquired.