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Alessandro Rabiolo, Fabio Bignami, Chiara Giacomini, Anna Lorusso, Giulio Ferrari, Paolo Rama; VesselJ: a new tool for automatic measurement of corneal neovascularization ex-vivo.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4352.
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© ARVO (1962-2015); The Authors (2016-present)
To describe and validate a novel automatic method (VesselJ) to quantify blood and lymphatic vessels in murine corneal flat-mounts.
Corneal neovascularization (CNV) was induced in 20 corneas of ten 6- to 8-week-old C57BL6/N mice by alkali burn (n = 10) and sutures (n = 10) assays. All corneal flatmounts were stained with CD31 and LYVE1. Three independent operators assessed blood and lymphatic CNV with both a quantitative manual (mCNV) and an automatic (aCNV) method. In the manual method the inner tips of the vessels were connected together. The resulting area was then normalized for the corneal surface and the result was mCNV. Automatic analysis was performed using a novel open-source plugin for ImageJ software, called VesselJ. VesselJ algorithm relies on a corneal background-adjusted threshold, based on the red channel for blood vessels and on the green channel for lymphatic vessels.
Both methods showed a strong reliability (ICC > 0.90) in quantifying hemangiogenesis for the suture and alkali burn models. However, reliability of lymphatic mCNV varied from moderate in the alkali burn (ICC: 0.700) to poor in the suture model (ICC: 0.415), whereas it remained high in aCNV. In the sutured group, a significant correlation between mCNV and aCNV was found among all three operators for blood vessels (P < 0.001) and just for one operator for lymphatic vessels (P < 0.001). Regarding the alkali burn model, correlation between blood mCNV and aCNV was significant for all operators (P < 0.001), whereas no significant correlation was appreciated for lymphatic vessels. Time spent to analyze images with VesselJ was significantly inferior to manual system (P < 0.001), being 6 and 21 times faster than manual method for blood and lymphatic quantification, respectively.
The mouse cornea is a generally accepted model to study blood angiogenesis and lymphangiogenesis and to investigate their role even in non-ocular disorders (e.g. cancer). The majority of the techniques used to quantify CNV relies on manual methods, thus being operator-dependent, time consuming and not always reproducible. VesselJ is a reliable and fast method to quantify corneal hem- and lymph-angiogenesis in corneal flat-mounts.
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