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Richard Foxton, Ashwath Jayagopal, Nadine Colé; Real-time measurements of vascular permeability in the mouse eye using vitreous fluorophotometry. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2030.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetic retinopathy (DR) is a leading cause of blindness, characterized by vascular hyperpermeability (VP), a key factor in vision loss. Animal diabetic models show some DR changes, including increased VP; however quantification often requires sacrificing animals (e.g. Evan’s Blue). We present here a method for detecting VP using vitreous fluorophotometry, enabling real-time measurements of fluorescent dye accumulation in mouse eye; including retina, vitreous and anterior chamber/cornea (AC/C), throughout the animal’s lifespan.
The Fluorotron Master Ocular Fluorophotometer Laboratory Mouse Edition (OcuMetrics, Mountain View, CA) with prototype eyelens was kindly loaned by OcuMetrics, Inc. Female 10-12 week old C57Bl/6J mice (Janvier, France), anaesthetized with Fentanyl 5 µg/kg, Medetomidine 0.15 mg/kg, Midazolam 2 mg/kg, and eyes dilated using 1% Tropicamide. Animals were injected with sodium fluorescein (NaF) intravitreally (IVT) for compartment localization, or intravenous (IV) for leakage assessment. To induce inflammation and leakage, lipopolysaccharide (LPS, 1ng) was injected IVT for 24 hr. Fluorescence was measured every 5 minutes, up to 50 minutes to profile NaF accumulation. Mice were recovered from anaesthesia by Naloxone 1.2 mg/kg + Atipamezol 2.5 mg/kg + Flumazenil 2.5 mg/kg. Data analysis was done with GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Analysis of Variance ANOVA (alpha 0.05) followed by post Hoc tests were performed.
IVT or topical 2ng or 20ng NaF enabled delineation of vitreous and AC/C. IV NaF at 0.25, 0.50 and 1.00mg caused dose-dependent fluorescence increase in retina (N = 4-6, P < 0.001), vitreous (N = 4-6, P < 0.01) and AC/C (N = 4-6, P < 0.05). In a time course, we observed a spike in retina fluorescence, followed by slow decline following an IV dose of 0.50mg NaF up to 50 minutes. This was accompanied by linear increases of vitreous and AC/C fluorescein. To test if the machine detected elevated VP accompanying inflammation, we injected LPS IVT, and observed a rapid elevation in vitreous fluorescein above vehicle (N = 5-6, P < 0.01)..
Vitreous fluorophotometry is a useful method for measuring VP in mouse eyes, and can be used over multiple time points, without sacrificing the animal. This method can be for basic research to study progression and factors underlying disease, but also for drug discovery and development.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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