Fig. 2

Retinal hypoxia and vascularization modifications after irradiation. (a) Hypoxic retinal areas are detected by the pimonidazole probe (in red) and the retinal vascularization is stained with lectin-FITC (in green) on rat flat-mounted retinas at one week, one month and six months after irradiation. Non-irradiated eyes were used as controls. Scale bars represent 1 mm. (b) Quantification of the retinal hypoxia intensity using the pimonidazole staining. One-way ANOVA statistical test followed by Tukey’s multiple comparison test, n ≥ 5 eyes. (c, d) Quantification of the surface covered by small and large vessels based on the lectin staining with a dedicated macro. After irradiation, we observed a significant and progressive decrease of small vessels and capillaries after irradiation along with a significant increase of larger vessels. One-way ANOVA statistical test followed by Tukey’s multiple comparison test, n ≥ 5, 13 images per flat-mounted retina were analyzed. * means p < 0.05; *** p < 0.0005; ****p < 0.0001. (e) Flat-mounted rat retinas stained with pimodinazole probe (in red) to lectin-FITC (in green) 6 months after irradiation. White squares show area enlarged at the bottom line. White arrows point the retinal microaneurysms. Scale bars represent 1 mm on merged and pimonidazole pictures, 100 μm on enlargements. (f) Transverse view of vessel blebbing in retina 6 months after irradiation. Vessel walls are stained with lectin in green and nuclei with Dapi in blue. All images represent the same vessel at different depth. L means lumen and white arrows show membrane blebs. Scale bar represents 10 μm