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Helder Andre, Monica Aronsson, Anders Kvanta; Mapping Angiogenesis on Choroidal Membranes of a Murine Laser-induced CNV Model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):336.
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In this study we investigate the expression and relative distribution of neoangiogenic molecules during the development of choroid neovascular (CNV) lesions in a laser-induced mouse model of neovascular age-related macular degeneration (AMD), with particular focus on the choroidal membranes of these lesions.
CNV lesions were induced on anesthetized 8-week-old C57BL/6 mice using diode laser photocoagulation. Animals were sacrificed in a time-course manner post-induction. For immunostainings, eye-cups were dissected from freshly enucleated eyes, and the choroids were shortly fixed. Choroids were immunostained for NG2 (pericytes), VEGF-R2 (vascular endothelial growth factor receptor-2; sprouting endothelial cells), or VEGF with co-staining for CD31 (vascular endothelial cells). Images of the choroidal membranes were obtained by fluorescence microscopy of the flat-mounts of the eye-cups. Vascularization was assayed by Evan's Blue perfusion on whole-eyes of laser-induced animals.
All choroidal membranes of the laser-induced CNV displayed positive staining for the four angiogenic markers studied. The relative distributions of the angiogenic markers were determined in comparison to the area of neovascularization, as observed by increased CD31 staining. Both NG2 and VEGF-R2 displayed a radial progression (inner to outer). NG2 matched closely the CD31 staining, while VEGF-R2 demonstrated a more demarked progression from the centre of the lesion to the outer limits through the time-course. VEGF displayed a uniform distribution across the choroidal membranes of the lesions in the studied times. Evan's Blue perfusion showed a discreet increase concomitant with the increase in vascularization observed by immunostaining throughout the time-course.
Our data indicates that the choroidal membranes of laser-induced CNV lesions recover through a canonical sprouting angiogenesis pathway, in response to a VEGF-mediated signal. In addition, these membranes display an increasing radial endothelial sprouting with concomitant pericyte recruitment from the laser injury to the exterior of the lesions, in a time-dependent manner. These results may have implications for the clinical understanding of CNV behavior in patients with neovascular AMD.
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