VesselJ: A New Tool for Semiautomatic Measurement of Corneal Neovascularization.

PURPOSE To quantify blood and lymph angiogenesis in mouse corneal flat mounts by means of a novel plug-in for ImageJ, called VesselJ, based on a dynamic threshold algorithm. METHODS Corneal neovascularization (CNV) was induced in the right corneas of 20 C57BL6/N mice by means of alkali burn (n = 10) or intrastromal sutures (n = 10). All corneal flat mounts were stained for blood vessels with CD31 and for lymphatics with LYVE1. Three independent operators measured blood and lymphatic CNV with both a published manual method (mCNV) and VesselJ (automatic method; aCNV). RESULTS Both methods showed a strong reliability, defined as intraclass correlation coefficient (ICC) > 0.90, in quantifying hemangiogenesis for sutures and alkali burn. However, reliability of lymphatic mCNV varied from moderate in alkali burn (ICC: 0.700) to poor in sutures (ICC: 0.415), whereas it remained high in aCNV (alkali ICC: 0.996; sutures ICC: 0.959). Among sutures, a significant correlation between mCNV and aCNV was found among all the three operators for blood vessels and just for one operator for lymphatic vessels (P < 0.001). In the alkali burn model, correlation between blood mCNV and aCNV was significant for all operators after excluding three noisy flat mounts (P < 0.001), whereas no significant correlation was seen for lymphatic vessels. CONCLUSIONS VesselJ is a semiautomatic, reliable, and fast method to quantify corneal hem- and lymphangiogenesis in corneal flat mounts. VesselJ can be easily used in the sutures model; it should be applied to other models (e.g., alkali burn) only after checking for background hyperfluorescence. Italian Abstract.