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S.H. Poor, C. Mailhos, W. Schubert, T. Adamis, S. Samuelsson, D.T. Shima; Novel Permeability Assay to Measure Retinal Barrier Function in Post–Natal Mice . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2997.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: One of the hallmarks of diabetic retinopathy is retinal damage due to increased edema caused by a breakdown of blood vessel permeability. The aim of this study is to better understand the nature of disruption of the blood–retina barrier. Studies of the blood–retina barrier have been hampered by a lack of suitable permeability assays which allow one to study the onset of this specialized vessel behavior. We have developed a novel permeability assay to monitor changes in vessel barrier function during mouse retinal development. Methods:FITC labeled 4 KD dextran was injected into the peritoneal cavity of neonatal mice. We first standardized the concentration, solution preparation, and volume injected per body weight. Eyes were enucleated at various times following injection, fixed, and imaged using Metamorph software. All parameters were identical and imaging was completed on the day of the experiment to avoid fading artifacts. Each retinal quadrant examined was given an arbitrary fluorescent unit value. Retinas from non–injected age–matched pups served as controls. The ratio of the average retinal fluorescence in the injected pup to the control pup was determined. Results:We examined the presence of FITC labeled 4–KD dextran in the retina of P6 and P16 pups at various times (1–15hrs) following injection to determine a time window for following a circulating tracer. We found that 2hrs post–injection best represented tracer localization in P6 and P16 mouse retinal vessels and proceeded to investigate vessel permeability to FITC labeled 4–KD dextran that had been circulating for 2hrs in P6, P9, P10, P12, P14 and P16 mice. P6 retinas examined 2hrs post FITC labeled 4–KD dextran injection had widespread leakage of tracer, and both fluorescence and retinal vessels were not delineated. At P9, there was weak, diffuse fluorescence in the peripheral extra–vascular retina, but the inner vessels were delineated suggesting heterogeneous barrier function. At P10 and later time points, there was minimal fluorescence leakage. Conclusions: We have developed an in vivo permeability assay using FITC labeled 4–KD dextran to study the onset of blood retinal barrier properties in neonatal mice. Restriction of a relatively small tracer from entering the extra–vascular space was observed by P10, a time associated with changes in the expression of permeability related proteins. We plan to use this assay to study vessel permeability in disease models, and for developing novel therapeutic strategies for treating ocular neovascular disease.
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