Spectral domain optical coherence tomography imaging

Subcutaneous injection of a mixture containing 67 mg/kg ketamine and 12 mg/kg xylazine was used for mice anesthesia. Pupils were dilated using tropicamide 1% eye drops (Ciclomidrin, Latinofarma, Sao Paulo, Brasil). Commercially available lubricants were used to prevent corneal drying (Systane, Alcon, Sao Paulo, Brazil).

SD-OCT images were acquired with the Heidelberg Retina Angiograph (HRA) Spectralis (Heidelberg Engineering, Germany). Three horizontal SD-OCT linear B-scans, centered on the optic nerve head, were obtained from each eye. SD-OCT scans were acquired in the automatic real-time mode (ART), averaging 30 frames per image, with a 30º covering field. The images with better quality were selected for analysis (Additional file 1). Retinal layer segmentation was performed automatically with the device internal software and manually corrected when necessary. The retina was segmented in: NFL + GCL (nerve fiber layer plus ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer), ONL (outer nuclear layer), IS/OS (photoreceptors inner segments and outer segments junction), and RPE (retinal pigment epithelium). Inner retina included NFL + GCL, IPL, and INL and outer retina thickness included OPL, ONL, IS/OS, and RPE (Additional file 1). Thickness measurements were performed 600 µm from the optic disc margin, nasally and temporally. The average values of total retina, inner, outer retina, and from each segmented layer thicknesses were used for analysis.