Comparison of intravitreal PBS injection timepoints in the mouse oxygen‐induced retinopathy model

Aims/Purpose: Intravitreally (IVT) administered phosphate‐buffered saline (PBS) is known to reduce neovascularization in the mouse oxygen‐induced retinopathy (mOIR) model, and often used as a vehicle control in preclinical efficacy studies. Here, the aim was to compare the effects of PBS on neovascularization and revascularization when administered at different timepoints. VEGF‐inhibitor aflibercept (Eylea®) was used as a reference control.

Methods: C57BL/6JRj male and female mice were exposed to 75 % oxygen from postnatal day 7 (P7) for 5 days. At P12 the mice were returned to normoxic conditions and IVT injected with PBS or aflibercept to the right eye at P12, P13, or P14 (n = 7‐9 eyes/group). Contralateral left eye was left untreated. Retinal flat mounts collected at P17 were stained with Isolectin B₄, and microscopy images covering the whole retinal flat mount were analyzed for the size of neovascular (NV) and avascular (AVA) area using a proprietary AI‐based algorithm.

Results: NV or AVA area did not differ between PBS and aflibercept at P12. Aflibercept reduced neovascularization by 83 % at P13 (Mann‐Whitney test, p < 0.001) and by 65 % at P14 (unpaired t‐test, p < 0.01) when compared to PBS, while AVA was similar between groups. When compared to untreated eyes, PBS significantly reduced neovascularization at P12, and the size of AVA area at P12 and P13 (Mann‐Whitney test, p ≤ 0.001).

Conclusions: Our findings show significant anti‐angiogenic effect of PBS when injected at P12, suggesting that P12 is not an optimal timepoint for IVT treatment if PBS is used as a vehicle. PBS administered at P13 or P14 did not block neovascularization while it still was inhibited by aflibercept. Further studies are needed to explore if similar effect at P12 is seen with other vehicle or neutral antibody controls.