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Yueying Liu, Kumar Sambamurti, Craig E. Crosson, Zsolt Ablonczy; RPE Barrier Dysfunction In A Rabbit Model In Vivo. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5648.
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Macular edema, the accumulation of fluid in the central subretinal space, is a major cause for the loss of visual acuity. The movement of fluid into the retina environment is controlled by inwardly directed hydrostatic and outwardly directed osmotic forces. To augment these passive forces, the RPE also has an active transport system to remove fluid towards the choroid. Disruption of the RPE barrier reduces the osmotic force and short circuits the active transport mechanism. Our goal was to develop a new model system to study the development and resolution of retinal edema.
Fluid leakage and transport across the RPE of Dutch-belted pigmented rabbits was measured by vitreous fluorescence (Oculometrics, Fluorotron Master), fluorescein angiography, and optical coherence tomography (Heidelberg Spectralis HRA-OCT). The fluorescence measurements were performed after injecting 25 mg/kg sodium fluorescein into the marginal ear vein. To test intravitreal VEGF-induced leakage, animals were treated 1 or 2 days prior the experiments with 5 µL intravitreal injections of VEGF (100 ng), the γ-secretase inhibitor DAPT (10 µM), or PBS. OCT experiments were performed with and without creating 1-5 µL volume subretinal blebs filled with above agents. The time course of the experiments was 0 - 180 minutes post fluorescein injection or bleb creation.
VEGF induced an increase in vitreous fluorescence, which was intensified with the co-administration of DAPT. Co-administration of VEGF and DAPT also resulted in fluorescence emanating from the avascular area of the rabbit retina (RPE leakage) compared to VEGF treatment alone. When examining the ZO-1 structure of RPEs from the same experiments, we found holes between the cells after VEGF administration but not in the controls. Reabsorption of PBS-filled blebs was biphasic with an initial rate of 0.94 V0/h, and final rate of 0.06 V0/h. VEGF-filled blebs reabsorbed slowly, at a rate of 0.03 V0/h).
Pigmented rabbits provide a useful model to study RPE barrier dysfunction, as the eye is relatively large and because of the mostly avascular nature of the neural retina. Therefore, fluorescence in the avascular areas signifies leakage through the RPE barrier. The use of OCT for the observation of subretinal blebs allows for accurate bleb size and reabsorption rate determination. These data demonstrate that VEGF disrupts the RPE barrier and transport functions in vivo, and that blocking γ-secretase activity with DAPT accentuates the effect of VEGF.
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