Quantification of vascular tortuosity as an early outcome measure in oxygen induced retinopathy (OIR).

Oxygen-induced retinopathy (OIR) in mice is a popular model system to study pathological angiogenesis in the retinal vasculature. The system is based on vessel depletion by exposure to hyperoxia, which results in acute retinal hypoxia upon return to room air. This hypoxia then triggers neovascularization in the remaining vessels after 5 days. Here we aimed to establish an additional and earlier experimental readout of the vascular response to hypoxia by quantifying the tortuosity of retinal arteries after 2 days. Mouse pups from three different mouse strains were exposed to hyperoxia from postnatal day (P) 7 to P12 and retinas were analysed at P12, P14 and P17. Hypoxia was assessed by staining with the hypoxia marker EF5 and by measuring Vegf mRNA by qPCR. The retinal vasculature was stained in whole mount retinas and tortuosity of radial arterioles was quantified. C57BL/6J mice were used because the vascular response at P17 is well characterised in this strain. We also used C3H/HeJ mice, which contain the retinal degeneration 1 (Rd1) mutation (Pde6b(Rd1)) and have abnormally thin retinas. These thinner, C3H/HeJ retinas do not become ischemic during the OIR model and do not develop neovascularization. They can therefore be used as a control. In addition, we included C3H/HeJ mice that lack the Rd1 mutation (C3H/He(Rd1-)), with normal thickness retinas, to control for strain differences between C57BL/6J and C3H/HeJ. Quantification of vessel tortuosity at P14 showed tortuous arteries in normal thickness retinas (C57BL/6J and C3H/He(Rd1-)) and straight arteries in the thin C3H/HeJ retinas. This correlated with hypoxia, which was severe in normal thickness retinas and mild in the thin C3H/HeJ retinas. Furthermore, at P17 the normal thickness retinas showed strong neovascularisation whereas in the thin C3H/HeJ retinas the retinal vasculature regenerated normally. In conclusion we have demonstrated that arterial tortuosity can act as an early readout for hypoxia in the OIR model before neovascularisation develops.