Diver 2

ONL, INL, PR, and FRT were measured using OCT (Bioptigen 840 nm; Phoenix-Micron, Inc., Bend, OR, USA). OCT images were obtained in WT, IRBP Het, and IRBP KO mice at 1 month postnatal. OCT collection and layer thickness analyses were performed using previously published methods [39 (link)], and 10 marks corresponding to specific reflective layers of the retina were placed using Bioptigen Diver 2.4 software. FRT was measured as the difference between marker 1 and 10 (Figure 1), corresponding to the inner limiting membrane and the outer edge of the RPE, respectively. The ONL was measured between markers 5 and 6, corresponding to the hyporeflective region above band 1. The INL was measured between markers 3 and 4, corresponding to the hyporeflective region above the ONL. Finally, the PR was measured between markers 6 and 9, corresponding to bands 2 and 3, which encompasses both the inner and outer segments, respectively. Measurements were determined at eight equidistant eccentricities in the nasal and temporal directions from the optic nerve head (±0.56, ±0.42, ±0.28, ±0.14). The average ONL, INL, PR, and FRT for all eccentricities were calculated and two-tailed t-tests were performed to determine differences between genotypes.

NOD-SCID mice (NOD.CB17-Prkdescid/J, Jackson Laboratories, USA, male, 4–5-weeks-old) were used for the study. A large sample size, n = 10, was taken to nullify any experimental anomaly. Mice were anaesthetized and sub-retinal injections of neutrophils from different experimental groups or recombinant LCN-2 protein were given as described earlier^{73 (link)}. Seven days after treatment, the NOD-SCID mice were anaesthetized by intraperitoneal injection of a ketamine and xylazine mixture and then subjected to Fundus imaging along with OCT analysis using the Bioptigen Envisu R2210 system. OCT images were analyzed on optical sections (100 sections per retina) from each eye ranging from−2.0 to +2.0 mm with respect to the optic nerve head (ONH) using the FIJI-ImageJ (NIH) plugin provided with the instrument along with Diver 2.4 software (Bioptigen). After the experiment, the animals were euthanized with CO₂ gas and the eyes were harvested for further experiments.