ABSTRACT
Background Choroidal neovascularization (CNV) is a main cause of visual impairment in many retinal diseases.To create an ideal CNV animal model is very important for the experimental and clinical study of CNV.The assessment method of repeatable and reliable for CNV model is still seldom.Objective This experiment was to explore the label value of fluorescent antibody for visualizing and quantifying the morphologic changes associated with laser-induced CNV.Methods Laser-induced CNV models were created in 30 eyes of 15 male SPF C57BL/6J mice by Krypton red laser irradiating fundus 2 spots around the optical disc with the wavelength 647.1nm,power 260 mW,spot diameter 50μm and exposure time 0.05 seconds.The CNV was evaluated at 5 minutes,4,7,14 and 28 days after laser injury by using fundus photography and fundus fluorescein angiography (FFA),and the successful models were identified as the rupture of Bruch's membrane.The mice were then immediately sacrificed and the eyeballs were enucleated to prepare the choroidal flatmounts.The posterior eye cups were fluorescently labeled with markers of cell nuclei (DAPI,4',6'-diamino-2-phenylindole),endothelial cells (isolectin-B4),and filamentous actin (phalloidin).The CNV areas from specimens were measured by Image pro plus 6.0.Two eyes from one matched mouse without receiving photocoagulation were used as the controlls.This study followed the Standard of Association for Research in Vision and Ophthalmology.Results No any CNV was seen in photocoagulated eyes in 5 minutes and 4 days after laser irradiation.The first sign of CNV appeared at 7 days following photocoagulation.The incidence of fluorescein leakage was 76.47% (26/34),81.81% (18/22),50.00% (5/10) at 7,14 and 28 days,respectively.The fluomicroscope examination showed that in unphotocoagulated areas,retinal pigment epithelial (RPE) cells were visualized with a uniform hexagonal array.Immediately after laser exposure,a circular area devoid of fluorescent labeling was observed,indicating disruption of the choroid-Bruch membrane-RPE complex.On the fourth day,cellular debris and fragmented nuclei were presented and an autofluorescent ring was visible at the site of Bruch's membrane disruption.The number of CNV vessels increased exponentially during the next 3 days.At 7 days,a well-defined isolectin-B4 labeled CNV network was exhibited and lasted for 28 days.The CNV areas were (7.99±0.42)×103μm2,(16.89±3.77)×103μm2,(14.37±4.02)×103μm2 at 7,14 and 48 days after photocoagulation respectively,showing a significant difference among these three groups (F=17.340,P=0.000),and the CNV area was significantly increased in the photocoagulating eyes in 14 days and 28 days compared with 7 days (q=16.46,q=15.54,P＜0.01).Conclusion Fluorescent antibody labeling allows the well identification and measurement of laser-induced CNV lesions in mouse choroid/RPE flatmounts.This technique offers excellent morphologic detail and facilitates the study of critical early events in CNV.CNV complexes are labeled at an early stage,providing a more accurate preclinical evaluation of antiangiogenic molecule.