Streampix 6

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).

OCT images were taken 1, 3, 7, 14, 30, 90, and 180 days after laser photocoagulation, as were corresponding OCT scans using a Micron IV fundus camera and an OCT Scan Head equipped with a mouse objective lens (Phoenix Research Labs, Pleasanton, CA, USA). The OCT device featured a broadband superluminescent diode at 830 nm, customized for retinal imaging of mice. The scan region on the mouse retina was 1.8 mm in the X and Y directions. Linear OCT scans consisted of a series of 1024 single point A-Scans. Right eyes had previously been dilated with 0.5% tropicamide (Santen Pharmaceutical Co. Ltd., Osaka, Japan) and hydroxyl ethyl cellulose (Senju Pharmaceutical Co. Ltd., Osaka, Japan), used as coupling gel. Images were captured from 20 positions for each eye using StreamPix 6 and Micron OCT commercial software (Phoenix Research Labs). Captured images were quantitatively analysed using “In Sight” software, which can automatically detect and measure each retinal layer. Using this software, the retinal thickness was measured at 20 recorded positions for each eye, and the average of all positions represented the overall retinal thickness.