Tj. Conrad et al., IN-VIVO MEASUREMENT OF CORNEAL ANGIOGENESIS WITH VIDEO DATA-ACQUISITION AND COMPUTERIZED IMAGE-ANALYSIS, Laboratory investigation, 70(3), 1994, pp. 426-434
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.