Quantitation of corneal neovascularization using computerized image analysis

We have developed a method for quantitating corneal neovascularization, induced in anesthetized rats by silver nitrate/potassium nitrate cauterization, using a LeMont OASYS video input image analyzer. Corneal vessels are visualized by perfusing the upper half of deeply anesthetized animals with a mixture of 10% india ink, 11% gelatin in lactated Ringer's solution. The eyes are then rapidly cooled using a stream of compressed dichlorodifluoromethane (Freon) to solidify the gelatin mixture. Magnified images of flat preparations of the corneas are acquired using a television camera attached to a dissecting microscope. The images are electronically converted to digital form and the digitized data are stored in the image analyzer. The area of the cornea and blood vessels are independently determined by analyzing the digitized data as discrete values of varying shades of gray (gray-scale analysis). The area and gray scale of the injury and its distance from the corneoscleral limbus can also be measured to determine variability of location and intensity of the injury in different animals. This technique allows the area occupied by new blood vessels during studies on corneal neovascularization in rats to be rapidly quantitated.