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Imran Ahmed Bhutto, Anupama Tiwari, Benjamin R Thomson, Malia Michelle Edwards, Gerard A Lutty; Visualization of choroidal vasculature in pigmented mouse eyes. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2235.
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The choroidal vasculature has been a recent focus of interest because of its involvement in AMD. The understanding of pathologic changes in the choroid requires an exact knowledge of the normal anatomy, especially the choroidal vasculature. Although the general distribution of the choroidal vasculature of primates has been described, the choroid vasculature of pigmented rodents has been a challenge. For significant assessment of choroidal vasculature in pigmented animals, the removal of melanin pigment is desirable. Here we report a method to bleach the melanin from choroidal melanocytes to visualize the choroidal vasculature that is compatible with immunohistochemical staining of the choroid.
After enucleation of eyes of pigmented C57BL/6J mice, retina was separated from the choroid, and the choroid was soaked in 1% ethylenediaminetetraacetic acid (EDTA) for 2 hours at room temperature to remove RPE. The choroid was fixed in 2% paraformaldehyde and subjected to melanin bleaching with 10% hydrogen peroxide (H2O2) treatment at 55°C for 90 minutes and immunostained for podocalyxin to label choroidal vessels. Images of the flat-mount choroid were taken using Zeiss 710 confocal microscope. This melanin bleaching method was also used to analyze the choroid from laser-induced choroidal neovascularization (CNV) and subretinal NaIO3 RPE atrophy animal models.
The H2O2 pretreatment effectively bleached the melanin, and made the pigmented choroid transparent. Immunolabeling was successful with the podocalyxin antibody and effectively labelled all layers of choroidal vasculature. In the posterior choroid, long posterior ciliary arteries formed several branches on each side supplying the adjacent choriocapillaris (CC). The CC appeared as a nonhomogeneous and nonlobular monolayer capillary network, consisting of dense honeycomb and irregular patterns. The venous blood vessles from the anterior choroid, and half of the posterior choroid drain into the vortex veins, one in each quadrant. Furthermore, this method also provided valuable insights into laser-induced CNV and demonstrated attenuation of CC posterior to the area of RPE loss in the NaIO3 model.
Pigment bleaching for staining of choroid whole mount allowed integral imaging of the choroid, which could not be accomplished with existing histological methods. Antigenicity of the antibody was also well preserved using this method.
This is a 2020 ARVO Annual Meeting abstract.
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