IN-VIVO MEASUREMENT OF CORNEAL ANGIOGENESIS WITH VIDEO DATA-ACQUISITION AND COMPUTERIZED IMAGE-ANALYSIS

BACKGROUND: Measurement of corneal angiogenesis is useful for quantita ting the effects of angiogenic stimuli and for evaluating the efficacy of potential inhibitors of neovascularization. Because accurate metho ds to record the entire pattern of corneal neovascularization over tim e in individual living animals do not exist, we have developed a nonin vasive method to achieve this goal. EXPERIMENTAL DESIGN: The technique couples video data acquisition methods with computerized analysis of the video images. A stereotactic holding and positioning device allows alignment of the cornea such that it is viewed in a known and repeata ble way. Contrast between blood vessels and corneal stroma in the imag es is enhanced by illuminating the cornea with monochromatic light cen tered on the peak absorption of hemoglobin. For each observation, mult iple overlapping images of the peripheral cornea are recorded on video tape and subsequently digitized with a computer image analysis system. Overlapping regions are found by either statistical cross-correlation or common object identification methods. A montage of nonoverlapping adjacent images is made. Background electronic signals are reduced and contrast is enhanced in each montage with the aid of image processing . Finally, vessel area is calculated by pixel counting after establish ing the density range for vessel identification. To demonstrate the ut ility of the method, we measured over the course of 18 days, the total area of neovascularization in rabbit corneas cauterized with silver/p otassium nitrate, and treated topically with either normal saline (con trol) or 1% prednisolone acetate (100 mu l four times daily for 10 day s). The corneal angiogenic response was measured at the time of cauter y and at selected intervals thereafter. RESULTS: The amount of angioge nesis in each control cornea increased progressively during the entire observation period. In contrast, the prednisolone-treated corneas man ifested less neovascularization than controls during the treatment int erval. After treatment ended, the amount of corneal angiogenesis incre ased slightly in this experimental group. This method provided multipl e data points from each animal. CONCLUSIONS: To date, accurate measure ment of corneal neovascularization: has been a time-consuming process yielding few data points for each animal studied. The new method descr ibed, accurately measures even small changes in the area of corneal ne ovascularization, and allows for multiple observations of the same ani mal. The technique as currently developed, however, is not applicable to corneas that are markedly opaque or associated with intracorneal he morrhage.