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M.M. Campos, R.N. Fariss, P. Becerra, J. Amaral; Novel Technique to Evaluate CNV Morphological Changes in RPE/Choroid Flat–Mounts by Confocal Microscopy . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3371.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have developed a novel confocal imaging technique for visualizing and quantifying morphological changes associated with laser–induced CNV, using rat RPE/ choroid flat–mounts. Methods: A Noedynium–doped Yttrium Aluminum Garnet (ND: YAG) 532 nM laser was used to induce Bruch’s membrane break, recognized by bubble formation. Eight burns per eye were applied. Rats were euthanized at 1, 4, 7 and 14 days after laser. Eyes were fixed, retinas removed, RPE/choroid labeled with fluorescent markers for nuclei (DAPI) endothelial cells (isolectin IB4) and filamentous actin (phalloidin) Specimens were flat–mounted and multiplane Z series were collected on a Leica SP2 confocal microscope to evaluate three–dimensional structures. Volocity Restoration& Classification modules (Improvision®) were used to deconvolute, analyze and quantify the lesion volumes from confocal Z series. Results: In non–lasered areas, RPE cells appear as a uniform hexagonal array. A honeycomb–like pattern of phalloidin labeling identified the circumferential actin bundles. Isolectin IB4 was restricted to the endothelial cells of the choroidal vasculature. Immediately after laser, there was a circular defect reflecting the disruption of the normal morphology of the choroid, Bruch’s membrane and RPE. At the center of the lesion, phalloidin and isolectin labeling were abolished and DAPI–labeled nuclei appeared severely disrupted. One day after laser, there was an extended area of coagulative necrosis with ghost–like choroidal vessels, cellular debris, fragmented nuclei and inflammatory cells. A circular defect with irregular borders was detected deep to the RPE corresponding to ruptured Bruch’s membrane. Two days post–laser, proliferation and migration of RPE and other cellular elements was apparent. Phalloidin labeling allowed visualization of actin stress fibers. By day 4, lectin–positive cells formed vessels in the injured area. By seven days, a well defined choroidal neovascular membrane was formed, which by 14 days was extended toward the sub retinal space Conclusions:Confocal microscopy allowed a three–dimensional reconstruction of CNV lesions and also permitted the evaluation of morphologic changes in each area.In addition of providing excellent morphological detail, this new technique permits quantification of the volume of the lesions in 3–D reconstruction. as well as, the role of early cellular signaling events, growth factors and inflammatory cells during CNV induction..
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