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Elliott H Sohn, Chunhua Jiao, Xiuying Liu, Weize Sun, Robert F Mullins; Visualization of mouse choroidal and retinal vasculature using fluorescent tomato lectin perfusion. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1252.
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© ARVO (1962-2015); The Authors (2016-present)
There is mounting evidence implicating choroidal vascular changes in the pathogenesis of AMD. Current methods to readily visualize this critical structure in mice present challenges as they result in incomplete labeling of choroidal capillaries, have nonspecific staining, or are outside the visible spectrum. We tested fluorescent DyLight-594 conjugated tomato lectin (TL;Lycopersicon esculentum agglutinin) to develop a reliable and simplified method to readily assess choroid and retinal vasculature on whole mount and cross sections in mice.
Adult albino mice, B6-TyrC/J (n=16) or BALB/C (n=10), were used in this study. After sedation, mice received 100-200mg (1mg/mL) of TL intravascularly through the tail vein (n=10), jugular vein (n=4), or left ventricle of the heart (n=12), followed by perfusion transcardially with 10mL of sodium phosphate buffered 4% paraformaldehyde (PFA). Eyes were enucleated and immersed in PFA for overnight fixation. Whole mounts of neural retina and choroid were prepared and evaluated using fluorescence microscopy. Co-labeling of choroidal endothelial cells on cross-section with FITC-conjugated Griffonia simplicifolia Lectin I isolectin B4 (GSL-IB4) or immunostaining with carbonic anhydrase IV (CA4) was performed. Percentage of perfused choroidal and retinal vessels were assessed semi-quantitively by three masked graders. A subset of eyes were subjected to thermal laser damage 24 hours prior to perfusion.
Intravascular injection of TL led to consistent and robust labeling of retinal and choroidal vascular walls. On cross-sections, labeling was co-positive for GSL-IB4; choroidal capillaries were co-positive with CA4. On flat mount, TL perfusion resulted in labeling of 99.4% (n=14) of choroidal vessels using tail/jugular vein injection compared to 84.9% (n=12) via cardiac perfusion (p<0.001). No difference was found between injection via tail/jugular vein vs cardiac perfusion in labeling of retinal vessels (94.2%, n=8 vs 99.9%, n=14, respectively; p=0.107). Vascular damage was detected easily using this method for eyes that underwent laser prior to perfusion.
Tomato lectin injection intravascularly can reliably label normal and ischemic choroid and retinal vasculature in mouse eyes in a quick and simple manner. This method should facilitate detailed study of retinal diseases such as AMD, diabetes, and other angiogenic processes in rodents.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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