Journal d'ophtalmologie clinique et expérimentale
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A Dual-Staining Method to Distinguish Retinal Vessels in Oxygen- Induced Retinopathy (OIR) Model

Ling Xu, Ping Hu, Ye Zhang, Jinyi Long, Xue Wu, Yuhong Ye, Ning Cui, Fei Sun, Xinmin Lu, Jikui Shen, Da Long

Purpose: The purpose of this study was to improve a time-saving, cost-effective, and labor-saving method to distinguish the retinal structural vessels, Neovascularization (NV), and Non-Perfusion (NP) areas in Oxygen-Induced Retinopathy (OIR) mice.

Methods: C57BL/6J mice were exposed to hyperoxic conditions (75% ± 2% O₂) from Postnatal day 7 (P7) to P12 and then returned to room air to cause maximal retinal Neovascularization (NV). After Retinas were isolated at P17, the retinal whole mounts were successively stained with Griffonia Simplicifolia Lectin (GSL) IB4-594 and GSL IB4-FITC using time- and concentration-controlling staining methods. This improved method only used two staining mediums and no other blocking mediums were required. The images were acquired by fluorescence microscopy and merged by Adobe Photoshop. Retinal neovascularization area and non-perfusion area were measured by Image-Pro Plus.

Results: Staining with the concentration of GSL IB4-594 at 1:100 for 45 minutes could almost completely stain all the neovascular tufts, which could assess the reliable and consistent status of retinal neovascularization. Meanwhile, the OIR retinal whole mounts were stained with the concentration of GSL IB4-FITC at 1:100 overnight followed by GSL IB4-594 staining allowed easy identification and quantification of retinal non-perfusion areas, structural vessels and neovascular tufts in the OIR mice.

Conclusion: The use of a reproducible and quantifiable retinal dual-staining to assess the NV and NP in the OIR model demonstrates the importance of an appropriate staining method that can easily calculate the ratio of NV and NP. Other results such as evaluating the anti-angiogenesis efficacy of bi-specific peptides for anti-angiogenesis obtained using this method suggest enhanced clinical and scientific research value that the ratio of NV and NP can be easily identified by using retinal dual-staining.