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Yan shao, Jianglei Chen, Xiaorong Li, Jian-Xing (Jay) Ma; Old Dog New Trick: Detection and Quantification of Retinal Neovascularization using BrdU Incorporation. Invest. Ophthalmol. Vis. Sci. 2020;61(7):181.
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The retina is a commonly used tissue for studying angiogenesis in nerve system due to its special characteristics. Oxygen-induced retinopathy (OIR) provides a useful model to study ischemia-induced retinal neovascularization(NV) and to develop anti-angiogenic therapeutics. However, a simple, accurate and less-subjective quantification method of neovascularization remains to be developed.
BrdU was daily intrperitoneally injected into oxygen-induced retionopathy (OIR) mice (75% oxygen, P7-P12) from P11 to the day before ehthanaisa. Anti-VEGF was intravitreally injected into treatment group on P14. Eyes were enucleated, fixed and stained by isolectin and BrdU antibody. The retinas were flat mounted and the NV was observed under a fluorescence microscope. Quantification was performed using imageJ(NIH) without extra plugin.
BrdU was dominantly incorporated into fast developing preretinal vessels under OIR condition and was specifically recognized by BrdU antibody lately, providing a specific fluorescence staining of the pathologic angiogenesis. The specific BrdU staining of newly divided vascular cells was observed at P14 and increased dramatically after P16, which was clearly observed and simply quantified using BrdU staining on top of isolectin. The therapeutic effect of Anti-VEGF treatment whcih prevented vascular cell division and pathologic NV development was clearly monitored by BrdU staining.
Compared with classic quantification methods using only isolectin B4 staining of the entire vascular network, BrdU labeling, allowed us to distinguish newly formed vessels from the preexisting vessels and to accurate quantify the newly formed vessels using the ImageJ program. It provided a useful and sensitive method to study retinal neovascularization and to evaluate therapeutic effect of medical interventions against pathologic angiogenesis.
This is a 2020 ARVO Annual Meeting abstract.
Quantification of neovascularization on the retina of OIR mouse using BrdU staining. a. IB4 (red) and BrdU (green) staining of OIR mouse retina. b. Quantification of BrdU(%) of the total vascular networks. c-h, the quantification strategy. c. an original image, d. the inverted red channel, e. the inverted green channel, f. “Regions of Interest” (ROI) created in imageJ based on vascular networks selection, g. the green channel overlapped on the ROI, h. measurement of the percentage neovascular area.
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