Abstract
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.
Original language | English |
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Pages (from-to) | 8199-8206 |
Number of pages | 8 |
Journal | Investigative Ophthalmology and Visual Science |
Volume | 56 |
Issue number | 13 |
DOIs | |
Publication status | Published - Dec 1 2015 |
Keywords
- Blood vessels
- Corneal neovascularization
- ImageJ
- Lymphatics
- Plug-in
ASJC Scopus subject areas
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience