A novel imaging technique for experimental choroidal neovascularization.
OBJECTIVE
Choroidal neovascularization (CNV) is the end point of several ocular diseases that lead to blindness. The authors developed an imaging technique for visualizing and quantifying morphologic changes associated with experimental laser-induced CNV.


METHODS
CNV was induced using laser energy to disrupt Bruch's membrane. Rats were euthanatized immediately after laser injury and at 1, 2, 3, 4, 7, 14, and 60 days. Nonlasered eyes were used as the control. Eyes were enucleated and fixed, and the posterior eye cups were fluorescently labeled with markers for nuclei (DAPI; 4',6'-diamino-2-phenylindole), endothelial cells (isolectin IB4), microglia (CD11b), and filamentous actin (phalloidin). FITC-dextran perfusion was compared with our technique. A confocal microscope was used to evaluate flatmounted specimens. Computer software generated three-dimensional reconstructions for qualitative and quantitative analysis of confocal image stacks.


RESULTS
In nonlasered areas, RPE cells were visualized as a uniform hexagonal array. Immediately after laser exposure, a circular area devoid of fluorescent labeling was observed, indicating disruption of the choroid-Bruch's membrane-RPE complex. One day after laser exposure, cellular debris and fragmented nuclei were present, and an autofluorescent ring was visible at the site of Bruch's membrane disruption. The ring correlated with bubble formation and CNV induction. Three days after laser injury, phalloidin-labeled RPE cells and isolectin-labeled endothelial cells increased significantly, reflecting cell proliferation and migration. By day 4, isolectin-positive cells forming vascular tubes were visualized. The volume of CNV vessels increased exponentially during the next 3 days. By 7 days, a well-defined isolectin-labeled CNV network was present, and its volume was preserved for several weeks. CNV volumes calculated on the basis of FITC-dextran perfusion were significantly lower than volumes obtained using lectin-labeled samples.


CONCLUSIONS
A novel imaging technique was developed that allows a three-dimensional reconstruction and measurement of laser-induced CNV lesions in rat choroid/RPE flatmounts. This technique provides excellent morphologic detail and facilitates the study of critical early events in CNV, including the rupture of Bruch's membrane and the formation of endothelial clusters before vessel formation. CNV complexes are labeled at an earlier stage and more reproducibly than with FITC-dextran perfusion, providing a more accurate preclinical evaluation of antiangiogenic molecules.
Campos
Mercedes
M
Biological Imaging Core, National Eye Institute, NIH, Bethesda, Maryland 20892-0703, USA.
Amaral
Juan
J
Becerra
S Patricia
SP
Fariss
Robert N
RN
D000199:Actins / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism / Q000473:pathology; D020256:Choroidal Neovascularization / Q000378:metabolism / Q000473:pathology*; D003911:Dextrans; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007211:Indoles / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008297:Male; D017628:Microglia / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal; D010590:Phalloidine / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000473:pathology; D051381:Rats; D011914:Rats, Inbred BN; D053675:Versicans
D000199:Actins; D015415:Biomarkers; D039481:CD11b Antigen; D003911:Dextrans; D005456:Fluorescent Dyes; D006023:Glycoproteins; D007211:Indoles; D037102:Lectins; C508324:Vcan protein, rat; C015219:fluorescein isothiocyanate dextran; D053675:Versicans; D010590:Phalloidine; C007293:DAPI; D016650:Fluorescein-5-isothiocyanate