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Eleni Karatsai, Michael B Powner, Silvia Dragoni, Patric Turowski; Measurement of acute vascular permeability and associated signaling in ex vivo rat retinae.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3221. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Vascular leakage and interstitial fluid accumulation occur as a result of breakdown of endothelial barriers and constitute vision-impairing features of diabetic macular edema. Current therapeutic strategies involve neutralizing vascular endothelial growth factor (VEGF), a key mediator of increased vascular permeability. However, since there are considerable limitations to the effectiveness of anti-VEGFs, there is need to understand mechanism of VEGF- and non-VEGF-induced permeability pathways in the retina as well as crosstalk between them. Here, we report an ex vivo preparation of rat retina which allows the measurement of acute vascular permeability and underlying signaling.
Rat common carotid arteries were cannulated and heads perfused with heparin and cardioplegic solution. Retinae were isolated and either processed for immunohistochemistry or flat-mounted for permeability measurements: First, the retinal vasculature was filled with sulforhodamine through injection of a radial retinal artery. Then the loss of dye (baseline and in response to 50 ng/ml VEGF-A) was measured by time-lapsed video microscopy. Pixel intensity measurements from acquired images were plotted against time and permeability changes were calculated.
The vasculature of stabilized, ex vivo retinae appeared normal for at least 1 h after isolation as judged by immunohistochemical staining of VE-cadherin and Claudin-5. Basal permeability in microvessels was measured at 0.65 ± 0.16 x 10-6 cm/s (n=6 vessels from 4 retinae). Superficial exposure of retinae to 50 ng/ml VEGF-A induced a 1.42-fold increase in permeability (p < 0.01). Tissue (intravitreal) VEGF-A stimulation led to specific phosphorylation of p38, hsp27, eNOS and paxillin in the microvasculature.
Baseline and VEGF-A-induced microvessel permeability measured in our ex vivo retinal preparation was in the range of previously published values. VEGF-A also induced activation of signaling pathway typically linked to endothelial permeability. Taken together our ex vivo retina model faithfully reproduced the microvascular response to VEGF-A, which suggests that it can be used to study mechanisms of acute permeability in a physiological setting and to test pharmacological inhibitors of retinal permeability.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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