Scanning Laser Ophthalmoscopic Imaging of Acridine Orange-Stained Leukocytes in the Blood Vessels and Tissues of Neovascularized Rat Corneas

Purpose: To image leukocyte adherence to the vascular endothelium and extravasation into surrounding tissues in neovascularized corneas using acridine orange, a selective DNA stain, in rats Methods: Corneal neovascularization was induced by placing two sutures in the cornea of one eye of 12 Long-Evans rats. One week later, acridine orange (4 mg/kg) was injected via the tail vein and the eyes were evaluated using a scanning laser ophthalmoscope (SLO) 3-5 minutes, 20 minutes, and one hour after injection. Images were recorded on videotape for further analysis. Results: Acridine orange staining showed adherent leukocytes in corneal vessels at 3-5 minutes post-injection. After 20 minutes, leukocytes were seen in the corneal vessels and in the surrounding tissues. At 1 hour post-injection, decreased number of leukocytes were visible in the tissues, but none were seen in the corneal vessels. Conclusion: Acridine orange effectively demonstrate the leukocyte/endothelial interactions (leukocyte adherence) in the blood vessel, as well as leukocyte infiltration of surrounding tissues in neovascularized corneas. This technique may have prognostic value in corneal disease in which neovascularization is the pathologic component.