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05, n = 5 vs. normoxia), the global vascular connectivity in hyperoxia-exposed retinas was significantly reduced (p less then 0.05, n = 5 vs. normoxia). Applying principal component analysis (PCA) for auto-segmentation of the vertical sprouts, we corroborated the obliteration of the vertical sprouts bridging the secondary plexuses, as evidenced by impaired vascular branching and connectivity, and reduction in vessel volumes and lengths (p less then 0.05, n = 5 vs. normoxia). Conclusion Coupling 3-D LSFM with topological quantification