Figure 4.

Cromolyn-mediated mast cell inhibition nullifies the asymmetrical distribution of nasal and temporal neovascularization. A: Schematic experimental design showing the time points of treatment administration in BALB/c mice with intrastromal sutures (suts.). Mice were treated with phosphate-buffered saline (PBS) or 2% cromolyn (3 μL/treatment). After 4 days, corneas were harvested for immunohistochemistry analysis of corneal neovascularization. B: Ocular surface tear wash (5 μL/wash) was collected at 0, 1, 3, and 6 hours following PBS or cromolyn treatment of corneas with sutures to measure tryptase, a mast cell activation marker. C: Representative slit-lamp biomicroscope images of corneas with sutures on nasal or temporal side following PBS or cromolyn treatment on days 2 and 4 after suture placement. D: Binary images (left side) and cumulative bar chart (right side) depicting the vascular density of corneal neovascularization. Slit-lamp images were converted into binary images, and vascular density as percentage area of the vessels in the total cornea was calculated using the vessel analysis plugin in ImageJ version 1.52v software. E: Representative immunohistochemistry micrographs (left side) and cumulative bar chart (right side) showing vessel area in nasal or temporal sutured corneas treated with PBS or cromolyn. Corneas were harvested on day 4 after suture placement and immunostained with CD31 (fluorescein isothiocyanate). ImageJ version 1.52v software was used to quantify the vessel area. Representative data from three independent experiments are shown, and each experiment consisted of four animals. Data are represented as means ± SEM (B, D, and E). ∗P < 0.05, ∗∗P < 0.01, and ∗∗P < 0.001 (t-test). Scale bar = 100 μm (E). Co., cornea; Conj., conjunctiva; SL, slit lamp.