Mmp2 deficiency affects corneal lymphangiogenesis induced by thermal cauterization. (A) RT-PCR analysis of Mmp2 expression in the cornea in baseline (D0) and at 7 or 14 days after cornea cauterization. The graph depicts the densitometric quantification of Mmp2 mRNA normalized to the 28S signal. MW corresponds to molecular weight. (B) Lymphatic vessels were identified by LYVE-1 immunolabeling on whole-mount cornea issued from Mmp2-deficient mice (Mmp2 KO) and WT mice. Left column: low-magnification images of the entire area (bars, 1 mm); middle column: higher magnification of the left-panel inserts (bars, 200 μm); Right column: highest magnification showing the tip of lymphatic buds (bars, 20 μm). (C-H) Computer-assisted quantification of lymphangiogenesis according to Blacher et al.²³  (C) Global analysis of lymphatic vessel distribution around the initial limbal vessel. (D-G) Additional parameters characterizing the vasculature were normalized to the total area of the cornea (density): area covered by neoformed vessels (vessel area density, D); total length of the vessels (vessel length density, E); number of vessels (end point density, F); number of bifurcations (branching density, G). (H) Results expressed as percentages of the control value reveal that Mmp2 deficiency affected all parameters characterizing vasculature complexity, but not the maximal capillary length (Lmax). In panel B, left and middle columns, tissues stained with Alexa Fluor 488 (Molecular Probes) were used mounted in Vectashield mounting medium (Vector Laboratories), and images were captured at room temperature with AH3-RFCA fluorescent microscope (Olympus) coupled to DP72 digital camera (Olympus), using CellA 3.3 acquisition software (Olympus Soft Imaging Solutions) and a 4× NA 0.16 SPlan Apo lens (Olympus). Whole-mount pictures were assembled from 6 to 9 pictures with Photoshop CS4 software (Adobe Systems). In panel B, right column, tissues stained with Alexa Fluor 488 (Molecular Probes) were used mounted in Vectashield mounting medium (Vector Laboratories) and excited using a multi-Argon laser. Panels are Z-stack average projections of Z-series pictures that were acquired at room temperature, on a TCS SP2 confocal microscope (Leica Microsystems) with a 2 μm step size using Leica Confocal 2.5 acquisition software (Leica) and a 20× NA 0.4 N Plan L lens (Leica).