4.5. Scanning Laser Ophthalmoscopy (SLO) and Optical Coherence Tomography (OCT) Measurements

SLO uses laser scanning microscopy to obtain images (fundus photos) of the retinal surface. OCT uses near-infrared light to obtain high-resolution two- and three-dimensional images within the retina. SLO-OCT measurements were performed using a commercially available system (Heidelberg Engineering Spectralis combined HRA+OCT, Heidelberg, DE), modified for use with animals (Medical Workshop, Utrecht, NL). Detailed methods are described elsewhere [35,36]. In short, animals were anesthetized with an intraperitoneal injection of a mixture of ketamine (22 mg/kg for animals <4 weeks of age; 65 mg/kg for animals ≥4 weeks of age) and xylazine (2.2 mg/kg for animals <4 weeks of age; 7.5 mg/kg for animals ≥4 weeks of age) diluted in 0.9% NaCl. The eyes were locally anesthetized using tetracaine-hydrochloride drops (1% w/v) and were dilated using tropicamide (0.5% w/v), and atropine (1% w/v) drops. Hylocomod drops were applied to maintain corneal hydration at all times. A contact lens (5.2 mm in diameter; Cantor-Nissel, Brackley, UK) was placed. The standard 30° field of view equipment set was used. Animals were placed on a custom-made heated holder, eyes were kept moist using Hylocomod eye drops, and body temperature was monitored. Imaging was done using the Eye Explorer software version 1.9.14.0 (Heidelberg Engineering, Heidelberg, DE). For fundus images, infra-red (820 nm) intensity was manually adjusted to prevent overexposure. OCT imaging was performed using a volume scan (57 frames, 786 A-scans, 30° × 25°, 61 scans, Δ120 µm, 8.8 scans per second). The reference arm was manually adjusted according to the manufacturer’s instructions. The follow-up function was used whenever possible to ensure accurate thickness profiles between time points. Frame analysis was done on a total of five single OCT scans and corresponding thickness profiles. The chosen scans were the crossing of the optic nerve (ON = 0), the middle superior section (ON + 10), the superior section (ON + 20), the middle inferior section (ON - 10), and the inferior section (ON - 20). Within each selected OCT scan, retina thickness was determined on 1 mm intervals, with a 0.5 mm minimum distance from the optic nerve. The thickness values were averaged, normalized, and shown ± the standard error.

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