Semi-automated quantification of total and avascular retinal area in the rat model of oxygen-induced retinopathy

Quantification of retinal and avascular areas in the rat oxygen induced retinopathy (OIR) model is traditionally performed using time-intensive manual methods that may be subject to inter-rater variability. We developed and validated two open-source, semi-automated Fiji (Fiji is Just ImageJ) macros to measure total retinal area (RA) and avascular area (AA) in rat 50/10 OIR retinal flat mounts. Concordance correlation coefficients demonstrated high agreement between macro-derived and manual measurements for RA (0.988), AA (0.985), and AA/RA (0.983), with minimal bias observed. While inter-user variability was higher for macro-based AA measurements compared to manual methods, averaging results from two users improved precision. These semi-automated tools reduce analysis time and training burden while maintaining strong agreement with gold standard measurements. Although limited by user input requirements and single-laboratory validation, these macros provide accessible, standardized methods for retinal quantification and have the potential to improve reproducibility and efficiency in OIR research.