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Ewa Kubala, Randall Mrsny, Laura Paneghetti, David Shima; Development of the Rodent Inner Blood-Retinal-Barrier. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5144.
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© ARVO (1962-2015); The Authors (2016-present)
The blood-retinal barrier (BRB) and blood-brain barrier (BBB) protect, respectively, retina and brain from substances circulating in the blood that may affect the homeostatic regulation of the delicate neural microenvironment. Postnatal development of the murine retinal vasculature provides the opportunity to explore mechanisms of BRB formation through the use of tracers and pharmacological manipulation, and may also provide information that is useful in understanding disorders characterised by BRB dysfunction.
To examine the onset of barrier function during post-natal vascular development, we modified a previously described in-vivo tracer assay (Poor et al, ARVO 2006). Fluorescent lysyl-dextrans were delivered into the retinal circulation by intraperitoneal cavity injection at different stages of postnatal retinal development. Animals were perfused, eyes fixed and the retinas flat mounted and the leakage of tracer from the vasculature was evaluated by epifluorescence microscopy and image analysis. The role of VEGF-A and MLCK signalling pathways in BRB formation was examined through immunostaining, Western blot and pharmacological blockade.
3kD fluorescent dextran freely extravasated from the retinal vasculature at post-natal day 5 (P5), but levels decreased at P8 and a complete barrier to the tracer was present by P10. Similar results were obtained with a larger 70 kD dextran and a small 341 Da cross-linkable - NHS-biotin tracers. Use of the cross-linkable tracer demonstrated that leakage prior to P10 occurred at the growing vascular front at the periphery, but not in the central retinal regions. Blockade of VEGF-A signalling hastened barrier formation at the growing vascular front, suggesting that the BRB can only fully formed following completion of the superficial vascular plexus and the concomitant down-regulation of VEGF-A levels. Acquisition of BRB function was also accompanied by a decrease in endothelial phospho-PAK and phospho-MLC levels, which is consistent with the decreased VEGF-A signalling. Lastly, MLCK inhibitor also hastened barrier function, suggesting a critical need to regulate the acto-myosin cytoskeleton during BRB formation.
Our findings provide a framework for the continue investigation of the inter- and intra-cellular changes required for BRB formation, and will hopefully yield insight into developing therapies to repair the abnormal barrier during neurovascular retinal disease.
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